Project Summary Bdellovibrio bacteriovorus is a predatory bacterium capable of killing nearly all Gram-negative bacteria (1). Given the global rise of antibiotic resistance, and dearth of new antibiotics discovered in the past 30 years, this predator is well positioned as an alternative to traditional antibiotics (2). To date, researchers have determined the bacterium's basic lifecycle; following prey attachment, B. bacteriovorus enters the periplasm where it degrades host proteins and nucleic acids to fuel replication, and subsequently four to six new progeny escape the dead cell (3). For many years B. bacteriovorus research has been hampered by a lack of genetic tools, and the genetic mechanisms of predation have only recently begun to be established (4). Unsurprisingly, 40% of its genome consists of uncharacterized hypothetical genes, likely owing to its unique lifestyle and distant relationship to model bacteria like E. coli (5). Furthermore, the genetic determinants for prey resistance and sensitivity remain unknown (6). The Camilli lab and collaborators have recently completed genetic screens on both predator and prey aimed at answering these questions. Using transposon insertion sequencing (Tn-seq) (7), we have identified six prey-associated pathways involved in predation of the bacterial pathogen Vibrio cholerae, and over 100 predator-associated genes in B. bacteriovorus required for replication on planktonic or biofilm V. cholerae and E. coli. We hypothesize that these genes and pathways are involved in specific stages of predation, and can be characterized through several assays. For each V. cholerae mutant we will determine (a) changes in predator attachment efficiency by fluorescence microscopy and flow cytometry, (b) changes in the killing rate by measuring prey fluorescence and viable counts over time, and (c) identify kinetics and alterations in the predator lifecycle by transmission electron microscopy. Furthermore, (d) we will determine whether predator chemotaxis is differentially required for hunting motile versus non-motile V. cholerae. For each B. bacteriovorus mutant, we will (a) validate its importance to predation by mini Tn-seq and complementation, (b) determine whether the mutant can kill its prey (and is thus defective in a later stage of the lifecycle) by viable count assays, and (c) determine whether each mutant is defective at prey attachment by coupling a mini Tn-seq screen and fluorescence- activated cell sorting (FACS). Identifying prey mechanisms of sensitivity and resistance will inform the use of B. bacteriovorus in the clinic, and characterizing predator genes may allow genetic engineering of strains with altered host specificity or strains better able to kill bacterial pathogens.